Cell Engineering and Molecular Pharming for Biopharmaceuticals

The Open Medicinal Chemistry Journal, 2008, 2, 49-61 49

1874-1045/08 2008 Bentham Science Publishers Ltd.

Open Access

Cell Engineering and Molecular Pharming for Biopharmaceuticals

M.A. Abdullah*,1, Anisa ur Rahmah1, A.J. Sinskey2 and C.K. Rha3

1Department of Chemical Engineering, Universiti Teknologi Petronas, Tronoh, Perak, Malaysia

2Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

3Biomaterials Science and Engineering Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts

02139, USA

Abstract: Biopharmaceuticals are often produced by recombinant E. coli or mammalian cell lines. This is usually

achieved by the introduction of a gene or cDNA coding for the protein of interest into a well-characterized strain of pro-

ducer cells. Naturally, each recombinant production system has its own unique advantages and disadvantages. This paper

examines the current practices, developments, and future trends in the production of biopharmaceuticals. Platform tech-

nologies for rapid screening and analyses of biosystems are reviewed. Strategies to improve productivity via metabolic

and integrated engineering are also highlighted.

INTRODUCTION

Findings in the 1950s that DNA is the molecule that en-

codes proteins, which in turn control all the cellular proc-

esses inside the organism, have provided the impetus for the

biotechnology era [1]. This has led to the advent of recombi-

nant DNA technology and hybridoma technology in the

1970s, which marks the birth of modern biopharmaceutical

development. As far as drug discovery and development is

concerned, this is a significant milestone as some molecules

are too complex and far too difficult to be extracted and puri-

fied from living materials, or synthesized chemically [2]. Genetic engineering provides an alternative means for the

production of therapeutic proteins through the use of bacte-

ria, yeasts, insect, animal and plant cells. The compounds

produced provide alternative therapies for serious life threat-

ening diseases such as cancer, viral infection or hereditary

deficiencies, and other untreatable conditions [1].

Various technologies have since emerged ranging from

the innovations in broad-based, rapid screening and mac-

roscale analyses, to the sophistication in the imaging, control and automation technologies. Also contributing to the rapid

progress are the innovations in gene therapies, antisense, cell

surface engineering and molecular diagnostics. The produc-

tion of biopharmaceuticals via recombinant technologies has

led to new, innovative products, as well as significant im-

provements in quality and yield of existing products. They

are better defined scientifically, with consistent quality and

are free from infectious agents due to stringent cGMP guide-

lines [2]. The industrial scale manufacturing of penicillin G

by the fermentation of mould Penicillium notatum in the

early 1940s is the early success story of the use of living

cells for drug production to combat infection by Staphylo-

coccus and other bacteria [3]. Since mid-1970s, large scale

*Address correspondence to this author at the Department of Chemical

Engineering, Universiti Teknologi Petronas, Tronoh, Perak, Malaysia; Tel.: +605-3687636; Fax: +605-3656176; E-mail: [email protected]

production of hundreds of therapeutic proteins such as insu-

lin, monoclonal antibodies, interferons or interleukins, have

been developed [1,2,4].

The world wide pharmaceutical market is estimated to

grow to $1.3 trillion by the year 2020 [5]. While chemical-

based drug continues to be the major source of drugs, the

world-wide biopharmaceutical market in 2003 is estimated

in the region of $30-35 billion, accounting for 15% of the

overall world pharmaceutical market [6]. Of these, plant-

derived drugs and intermediates account for approximately $9-11 billion annually in the USA [2,5]. It was the scientific

and technological innovation in drug discovery and devel-

opment that had led to the creation of hundreds of start-up

biopharmaceutical companies in the 1970s and 80s. With the

basic research done in the universities and research institu-

tions, the synergies between industrial players and academia

over the years have resulted in the new technologies and

tools to find new molecules to combat diseases; development

of methods and biomarkers for clinical phenotyping; and

validation of biochemical hypothesis of a drug candidate

[1,2,4]. The growing confidence and interest in biopharma-ceuticals has pushed big pharma companies to acquire tech-

nologies or invest in manufacturing facilities. Merck has

bought RNAi developer, Sirna Therapeutics for $1.1 billion

(RNAi being short interfering molecules to inhibit any gene

of interest in any cells) [7]. Genentech has invested $140

million to set-up microbial-based manufacturing operations

for biotherapeutics in Asia [8]. Despite high expenses in

R&D, Merck and Genentech earn $32.8 and $1.4 billion in

revenue, respectively, in the year 2000 [4]. This review arti-

cle examines the practices, developments, and future trends

in the production of biopharmaceuticals.

HOST SYSTEMS FOR MOLECULAR PHARMING

The triggering factor behind the revolution in biopharma-

ceutical industries can largely be attributed to the develop-

ment of advanced methods in the field of recombinant DNA

50 The Open Medicinal Chemistry Journal, 2008, Volume 2 Abdullah et al.

technology. Cell engineering and transgenic technology bor-

der on several enabling techniques in such diverse fields as

cell biology, embryology, molecular genetics, bioprocesses

and metabolic engineering. A more directed approach to

improve the cells or a given pathway of interest have become

possible with specific genetic perturbations through modifi-

cation of the promoter strength of a given gene, or by gene

deletions, or by introducing whole new genes or pathways

into the cells [9-12]. This means that the alteration effects can be determined and the amount directly-probed and pro-

duced at a specified quantity.

Scientific advances gained by transgenic capabilities also

enable further understanding of basic biological pathways

and yield insights into how changes in fundamental proc-

esses can perturb programmed development or culminate in

disease pathogenesis [13]. Primary step behind recombinant

DNA technology is the introduction of heterologous gene(s)

in a non-native genetic background, and the sufficient ex-

pression of a cloned gene in the new host system. Two types of gene library are known, namely the genomic library which

includes all the total chromosomal DNA of an organism; and

cDNA library which corresponds to the mRNA fraction from

a cell or tissue at specific point and time [14].

Recombinant Microorganisms

Biopharmaceuticals produced from microorganisms that

have gained marketing approval are invariably produced in

the recombinant E. coli cell systems such as E. coli K12. The

species are well-studied, documented and optimized as hosts

for gene cloning [15-17]. These biopharmaceutical products

include tissue plasminogen activator, insulin, , -interferons,

interleukin-2, granulocyte colony stimulating factor and hu-

man growth hormone [1,2,18]. The advantages that are nor-

mally associated with E. coli as the source of biopharmaceu-

ticals include the well-characterized molecular biology and

the ease of genetic manipulation; high levels of heterologous

protein expression (as high as 25% of total cellular protein in

the case of -interferon); relatively simple and inexpensive

media, supported by well-established fermentation technol-

ogy [2].

However, vast bulk of heterologous proteins from E. coli

are intracellular which could complicate the downstream

processing. There is also the possibility of the formation of a

highly-densed inclusion body or insoluble aggregates of pro-

teins, which could overload the normal protein-folding mechanism. Being a gram negative bacteria, the presence of

endotoxin molecule lipopolysaccharides (LPS) in E. coli is a

concern [2,19]. LPS, which make up 75% of E. coli’s outer

membrane surface, influence the hypothalamic regulation of

body temperature when they enter the blood-stream, and

cause fever, which in some instances fatal. E. coli also often

does not recognize the upstream elements of genes derived

from different bacterial genera or families, or in some cases

the over expressed protein may be toxic. Yeast protein

kinase over-expressed in E. coli for example has been found

inactive, but the same protein is active when expressed in yeast [20]. It is therefore common to transfer a gene which is

originally cloned in E. coli, back into its native genetic back-

ground such as the -amylase gene cloned in E. coli but

transferred and expressed in its native Bacillus amylolique-

faciens, or antibiotic biosynthetic genes in Streptomycetes

species [2].

Many therapeutically useful proteins, when naturally

produced in the body are glycosylated. The limitation in bio-

logical activities of some expressed proteins from prokaryo-

tes has been attributed to their limited capability to carry out post-translational modifications (PTMs) [2,21]. Vast major-

ity of therapeutic proteins undergo several PTMs, which are

the final steps in which genetic information from a gene di-

rects the formation of a functional gene product. PTMs in-

clude covalent modifications of individual amino acids resi-

dues such as glycosylation, phosphorylation, methylation,

ADP-ribosylation, oxidation and glycation; proteolytic proc-

essing and non-enzymatic modifications such as deamidation

and racemisation. Most therapeutic proteins require at least

proteolytic cleavages, oligomerization and glycosylation for

their bioactivity, pharmacokinetics, stability and solubility

[22,23]. Polyglutamylation is an example of PTM that gener-ates lateral acidic side chains on proteins by sequential addi-

tion of glutamate amino acids. This modification is first dis-

covered on tubulins, and is important for several microtubule

functions [24]. As complex therapeutic proteins produced in

prokaryotes are not always properly folded to confer the de-

sired biological activity, microbial expression system is suit-

able mainly for the expression of relatively simple proteins

which do not require folding or PTMs to be biologically ac-

tive. Table 1 shows the comparison between different trans-

genic systems for the production of recombinant proteins.

Eukaryotic Cell Systems

Major advantage of eukaryotic expression systems such

as yeast, Chinese hamster ovary strain K1 (CHO-K1) or

Baby hamster kidney cells (BHK), is their ability to carry out

PTMs of protein product. While bacteria and yeasts may

only be suitable for the synthesis of antibody fragments, in-

sect cells infected by baculovirus and CHO cells can be the

source of intact antibodies. Animal cell cultures are being

used for the production of monoclonal antibodies via hybri-

doma cell technology; and vaccines production such as yel-

low fever viral particles via chick embryos culture, hepatitis

A viral vaccines via human diploid fibroblast or gp120 [2]. Fungi such as Aspergillus niger and yeast such as Pichia

pastoris have received considerable attention due to their

potential for high level of protein expression such as factor

VIII, -interferon or Human Serum Albumin; and protein is

excreted out into the extracellular media [25-27]. The gp160

HIV vaccines for example have been produced not only in

insect or CHO cell-lines, but also in Saccharomyces cere-

visiae [2]. Large scale production of polypeptides normally

present on the surface of pathogen can now be produced by

producer organisms such as hepatitis B surface antigen

(HBsAg) genes expressed in yeast for the production of clinically-safe subunit vaccines [28].

Yeast has received considerable attention due to its more

detailed genetics and molecular biology that can facilitate

genetic manipulation, and a long history of application in

brewing and baking. It is thus considered as GRAS organism

(Generally regarded as safe) [2,29,30]. Important advantage

Cell Engineering and Molecular Pharming for Biopharmaceuticals The Open Medicinal Chemistry Journal, 2008, Volume 2 51

for the use of yeast as host system is its ability to assemble DNA fragment in genomes by homologus recombination

which allows the insertion of DNA sequences at specific

locations in the yeast genome [31]. In the application of

“biodrug” into the gastrointestinal tract, yeasts can be advan-

tageous over bacteria, for the functional expression of het-

erologous genes, and they are not sensitive to antibiotics

with high level of resistance to digestive secretions [30,32].

A “biodrug” concept involves the use of recombinant micro-

organisms as new delivery vehicles which may have poten-

tial medical applications in the correction of enzyme defi-

ciencies, the control of the activation of pro-drug to drug or the production of therapeutic proteins, such as vaccines, di-

rectly in the digestive tract. The recombinant cells may pro-

duce active compounds such as hormones, enzymes, and

vaccines; or perform bioconversions or “biodetoxication”

[32,33].

The eukaryotic cell-based system however may need a

more complex nutritional requirement as compared to

E. coli. It may also require a carefully controlled-fementation

condition due to the shear sensitivity of the cells, and also to

control protein glycosylation which may depend on cell me-tabolism such as that in CHO [34]. Cell growth are much

slower, the post-translational glycosylation pattern especially

in yeast and fungal cells may be inappropriate or different

from the pattern observed in native glycoprotein isolated

from natural sources, and the cells are often proned to con-

tamination by virus or prions [2]. Scaling-up of cultured

mammalian cells to large volumes is more difficult. It may

take 4 years to build a 100000L fermenter, costing $400 mil-

lion for CHO cells [35]. Because of huge capital costs, in-

dustry has been unable to keep up with the growing demand

[36]. Another method that can be explored is to extract bio-pharmaceuticals from animal and human tissues such as in-

sulin from pig and cow pancreas, or blood proteins from

human blood [37]. However, these also incur high-cost and carry the risk of transmitting infectious diseases to humans

[38].

Transgenic Animal

The biochemical, technical and economic limitations of

prokaryotic and eukaryotic expression systems have spurred

interest in transgenic animal and plant as new expression

systems. Transgenic animal as a bioreactor system for phar-

maceutical production, or for modification of tissues and

organs for transplantations, or as a model system from DNA

microinjection to gene targeting and cloning, has had a sig-nificant impact on human health, pharmaceutical discovery

and the drug pipeline [13]. Transgenic modifications, par-

ticularly in mice, are commonly used to model human condi-

tions. The use of transgenic animal have been beneficial in

the studies for drug discovery in human developmental and

pathological conditions, including gene therapy, genetic ba-

sis of human and animal disease, the assessment of the valid-

ity of therapeutic strategies before clinical trials, disease re-

sistance in humans and animals, drug and product testing or

toxicological screening, and novel product development

through molecular pharming [13,39].

The production of therapeutic proteins from transgenic

animals involves the expression from mammary-gland spe-

cific promoters to drive secretion of the transgene into milk,

or the use of kidney- or bladder specific promoters that direct

transgene expression to the urine [40-42]. Mammary specific

expression is achieved by fusing the gene of interest with

promoter containing regulatory sequences of a gene coding

for a milk-specific protein, such as whey acid protein pro-

moter in the -casein and - -lactoglobulin genes [2]. The

early success story of transgenic expression of a Human Tis-sue-Type Plasminogen Activator (tPA) has been reported in

mouse milk [43] and Goat Milk [44]. The European Medi-

Table 1. Transgenic Systems for the Production of Biopharmaceuticals

Bacteria Mammalian Transgenic Animal Transgenic Plant

1. Productivity

g/L culture

2. Advantages

Easy to culture

Well-characterised

3. Limitations

Lack of PTMs

Limited capacity

4. Issues

Presence of inclusion bod-ies, endotoxin molecules

g/L culture

PTMs mechanism

Inappropriate PTMs

More complex media

Slow cell growth

Limited capacity

Viruses and prions contamination

g/L milk or urine

Low capital cost

Easy to harvest

Easy to scale-up

PTMs mechanism

Inconsistent product yield

Methods for genetic modification

kg/ha or

g/L (for latex)

Low cost

Easy to harvest

Easy to scale-up

PTMs mechanism

Low product yield

Environmental concern on GMOs

Need stronger promoters Targeted protein expression in spe-

cific organs, cell compartment


cines Agency (EMEA) has approved the use of ATryn, a

drug extracted from the milk of goats engineered to carry a

human gene involved in inhibiting blood clots [45]. For

monoclonal antibodies, the vast majority of production

source are of murine origin [2]. Major problems associated

with murine antibodies include the reduced stimulation of

cytotoxicity and the formation of complexes after repeated

administration, resulting in mild allergic reactions and

anaphylactic shock [46]. Rabbit milk is seen as an attractive alternative source for antibodies, as rabbit is not susceptible

to prion diseases and is known to transmit only rare and mi-

nor diseases to human [47]. Other various end-organs and

system being investigated for antibodies production include

blood, urine and other tissues, and egg white from transgenic

chicken. In 2002, a mature and functional human immuno-

globulins has been reported in the blood of transchro-

mosomic calf. This is achieved by introducing a human arti-

ficial chromosome (HAC) vector containing the entire unre-

arranged sequences of the human immunoglobulin (hIg)

heavy-chain (H) and lambda ( ) light-chain loci, into bovine

primary fetal fibroblasts using a microcell-mediated chromo-some transfer (MMCT) approach [48]. The use of the tran-

schromosomic calves is an important step in the develop-

ment of a system for producing therapeutic Human poly-

clonal antibodies (hPABs).

In the area of human xenotransplantation, the transgenic

models remain a viable option in dealing with severe donor

organ shortages. Research continues to address the biological

barriers with regards to hyperacute rejection mediated by

preformed natural antibodies and complement [13,49]. An important development in the area of xenotransplantation is

the stem cell and nuclear transfer cloning procedures such as

that being developed in the production of -1,3-

galactosyltransferase knockout pigs [50-52]. The embryonic

stem-cell technology has moved from the well studied trans-

genic mouse to now include the transgenic fish, chicken,

rabbits, sheep and cattle [53,54]. The main advantages of

transgenic animal as a source of biopharmaceutical produc-

tion are as shown in Table 1. High protein expression level is

achievable (in many cases exceeding 1 g protein/litre milk,

which may be similar to 50-100 litre bioreactor in a day) [2]. There is less environmental concern as transgenic farm ani-

mals are kept in enclosed areas. The only drawback is the

variability in the expression levels ranging from 1 mg/L to 1

g/L. This can be improved through vector optimization and

the use of gene insulators for increased and more predictable

production such as for antibodies in milk [47,53].

Transgenic Plant

Both transgenic animal and plants may be the only tools

capable of producing high level of protein or antibodies [47].

Different types of therapeutic proteins such as blood and

plasma proteins, vaccines, hormones, cytokins and growth factors, enzymes and others such as hirudin, endostatin and

human lactoferrin, have been produced in transgenic plant

systems mainly in tobacco and potato [22]. Many antibodies

or antibody fragments have been produced for therapeutic or

diagnostic purposes in various plant expression systems [55].

These plant-based antibodies are correctly assembled, prote-

olytically matured and glycosylated, with high mannose and

biantennary complex type N-glycans [56,57]. While animal

and plant cells may have similar capacity to assemble anti-

body subunits, plants may differ from animals in carrying

out PTMs, as far as the capacity to glycosylate antibodies is

concerned. This is pertinent as glycosylation is required for

stable antibodies in vivo and in inducing complement and

antibody-dependent cellular cyotoxicity (ADCC) [47]. De-

spite differences in N-glycan structures, antibodies produced in plants have similar antigen-binding capacity as their ho-

mologs produced in mammalian cells. But unlike mammal-

ian cell cultures, plants are devoid of human infective viruses

and prions [2,22]. Furthermore, an antibody half-life in the

bloodstream as well as its ability to be recognized by Fc re-

ceptors, which are both determined by heavy chains N-

glycosylation, are not strongly affected when a plant-N-

glycan is present instead of a mammalian N-glycan [58-60].

Glycosylation of antibodies can be improved in plants and

animals by transferring the genes encoding enzymes capable

of adding N-acetylglucosamine (GlcNac), sialic acid, fucose

and galactose to the N-glycans [47].

Current limitations of plant expression systems are low

yields of some therapeutic proteins and the impact of non-

mammalian glycosylation on the activity, immunogenicity

and allergenicity of glycosylated plant-made pharmaceuti-

cals. The N-glycan of antibodies extracted from plants (plan-

tibodies) have been reported to not only unable to confer

some biological properties and to induce immune response

when tested in mice, but also may have undesirable side-

effects in patients [47]. Different strategies and new plant expression systems are currently being developed to improve

the yields and quality of plant-made pharmaceuticals. The

challenges include in choosing the transformation systems,

adaptation of codon usage, gene silencing, design of recom-

binant transgenes with appropriate expression, tissue speci-

ficity and proper developmental regulation, and subcellular

localization of products [22]. The location of protein accu-

mulation within the cell is important to ensure correct fold-

ing and stability of the protein [61]. Different plant organs

(leaves, seeds, root) and plant cell compartments (endoplas-

mic reticulum, chloroplast, vacuole and oil body) have been used to express many therapeutic proteins [62,63]. In plants

with large foliage volume such as tobacco, alfalfa and leg-

ume plants, expression is performed in leaves. In potato,

corn, rapeseed, safflower, soybean, wheat or rice, protein

accumulation is achieved in tubers or in seeds [64,65]. In

plants, genetic material is distributed between the nucleus,

plastids, and mitochondria. Traditionally, the production of

transgenic plants, for basic and applied purposes, has been

mainly through transgenes expression in the nucleus. None-

theless, there is a concern that transgenes may escape and

contaminate the environment via pollen such as in corn [66].

There is now an increasing interest in expressing the trans-genes in chloroplasts rather than the nucleus as the genes

expressed in the plastome will not be transferred through

pollination [66,67].

Most biopharming applications target production and

storage in seeds, which naturally accumulate high concentra-

tions of proteins and oils, and the easiest part of the plant to


store and transport to processing facilities. Two seed-specific

“promoters” have been used experimentally - the beta-

phaseolin promoter of common bean and the oleosin pro-

moter of Brassica species [61]. Recent advances in the con-

trol of post-translational maturations in transgenic plants will

allow them to perform human-like maturations on recombi-

nant proteins and make plant expression systems suitable

alternatives to animal cell factories [22]. Antibodies that

currently cost thousands of dollars per gram might be pro-duced in plants for $200 per gram [68]. Transgenic plants are

expected to produce up to 10 kg antibodies per acre [69] or 1

kg of plantibodies after 36 months. This may be achievable

with rabbit milk, but not goat milk [58]. A plant “bioreactor”

will allow the production of recombinant proteins up to 20

kg/ha, regardless of the plant material considered – tobacco,

corn, soybean or alfalfa [58]. With the whole gamut of issues

pertaining to potential host systems for recombinant protein

production elaborated and recognized, in the next part, new

development and future direction of research in biopharma-

ceuticals production are discussed.

SCIENTIFIC AND TECHNOLOGICAL INNOVA-TIONS IN BIOPHARMACEUTICALS

Human Stem Cells

The discovery that cells are capable of self-renewal has

led to the functional definition of stem cells [70,71]. This has

great impact in the area of targeted therapies and drug

delivery as human stem cells may not only find application

in the repair, regeneration and cellular replacement of dam-

aged or defective tissues, but also in the toxicological screen-ing and discovery of new therapeutic drug molecules, and as

a tool for in vitro investigation of cellular and developmental

processes [72-74]. Human stem cells can be isolated, puri-

fied, expanded in number and differentiated into the cell type

of choice in a controlled manner. The cells may be sourced

or derived from blood and tissues postnatally (‘adult’ stem

cells), and from the fetus (fetal stem cells) or from the blas-

tocyst in the developing embryo prior to implantation (em-

bryonic stem cells) [72,75]. Adult stem cells and progenitor

cells found in adult tissues, act as a repair system for the

body, replenishing specialized cells, and also maintain the

normal turnover of regenerative organs, such as blood, skin or intestinal tissues. They have been used to treat

successfully leukemia and related bone/blood cancers

through bone marrow transplants [76].

Of great interest today is in the derivation and culture of

Human embryonic stem cells (hESC). These cells are pluri-

potent and undifferentiated, and can grow in vitro indefi-

nitely. They can potentially provide a supply of readily

available differentiated cells and tissues of many types to be

used for therapeutic purposes, as well as for drug screening and discovery [77]. The actual methods of hESC derivation

have not changed greatly since the method first porposed

[72,77]. It requires establishment of embryonic stem cell

lines using the inner cell mass of an early pre-implantation

embryo or excess human embryos from in vitro fertilization

treatments. To enable the clinical use of hESC for cell trans-

plantation, the use of animal derived biological components

is no longer acceptable. The main emphasis over the last

several years has been in finding defined conditions for deri-

vation and culture of hESCs. The aim is to replace even hu-

man derived materials with completely defined systems. The

use of embryonic stem cells is embroiled with ethical issues

as the blastocyst may be destroyed during the process

[72,77,78]. There are also challenges to generate cells of

sufficient quality and quantity and to expand cell numbers

while maintaining the fidelity of phenotype. Strategies need

to be developed to control and direct differentiation to pro-duce the cell type of interest in a format that is suitable for

intended purposes [79].

Gene and Targeted Therapies

Gene therapy is a novel technique, emerge as a direct

result of recombinant DNA revolution. Though still highly

experimental, it has the potential to become an important

treatment regimen as it allows the transfer of genetic infor-

mation into patient tissues and organs for the diseased genes

to be eliminated or their normal functions rescued. The pro-

cedure allows the addition of new functions to cells, such as

the production of immune system mediator proteins that help to combat cancer and other diseases [80]. The technique en-

tails stable introduction of a gene into the genetic comple-

ment of a cell, such that subsequent expression of the gene

achieves a therapeutic goal. The desired gene can be naked

DNA as in the case of DNA-based vaccine; or packaged into

a vector system such as retroviruses or plasmid-containing

liposomes, or microencapsulated, to affect gene transfer

[2,81-83]. Once assimilated by the cells, the exogeneous

nucleic acids must be delivered to the nucleus. The in vitro

approach necessitates removal of the target cells such as

blood cells, stem cells, epithelial cells, muscle cells or hepa-tocytes from the body, cultured in vitro together with vector

containing nucleic acid to be delivered, and the genetically

altered cells reintroduced into the patient’s body. Another

approach is direct administration of the nucleic-acid contain-

ing vector to the target cell, in situ in the body, such as direct

injection of vectors into a tumour mass, or aerosol admini-

stration of vectors containing cystic fibrosis gene to respira-

tory tract epithelial cells. For intravenous injection, vector

can be designed to be bio-specific such that it will recognize

and bind only to the specified target cells. Selective delivery

can be made possible by the inclusion of antibody on the vector surface, which specifically binds a surface antigen

uniquely associated with the target cell; or vector engineered

with a specific hormone that can only bind to cells display-

ing the hormone receptor [2]. The therapeutic potential of

gene therapy includes curing in-born errors of metabolism,

or conditions induced by the presence of a defective copy of

a specific gene (s), including cancer, AIDS, cystic fibrosis,

haemophilia, neurological disorder, or retinal degeneration

[2,84-86].

The antisense approach is a type of gene therapy based upon the generation of short, single-stranded stretches of

DNA or RNA, termed antisense oligonucleotides, displaying

specific nucleotide sequences. These oligonucleotides can be

synthesized and are capable of binding to DNA at specific

gene sites or mRNA derived from specific genes. The trans-

lation of mRNA can be blocked, preventing the synthesis of


mature gene protein product. This may have potential appli-

cation in treating disease states which require blocking of

gene expression for curing effect [2]. However, antisense

DNA technology has failed to live up to its early hype, as

most standalone antisense companies folded [7]. The down-

fall of antisense has been attributed largely to its off-target

effects, especially the tendency of nucleic acid sequences to

induce generalized immune responses, as well as the diffi-

culty of delivering the therapies to the right cell types.

Another nucleic acid-based therapy, short interfering

RNA (siRNAs) or microRNAs (miRNAs) or RNAi thera-

pies, have been hailed as the hallmark of new frontiers in

biotechnology [7]. These are small noncoding RNAs that

regulate gene expression by repressing translation of target

cellular transcripts. Though the extent of miRNA regulation

is not well known, increasing evidence indicates that this is a

naturally occurring mechanism in eukaryotes, and miRNAs

have distinct expression profiles and play crucial roles in

numerous cellular processes. miRNAs can induce a cell to destroy complementary pieces of mRNA, preventing the

target message from being transcribed. This RNA inhibition

can be exploited to inhibit any gene of interest and may be

particularly useful in gene therapies [2,7,87]. The major

challenges with developing RNAi therapies also involve

delivery to the target site, and cross-talk in signaling path-

ways. Transgene expression can be suppressed in hema-

topoietic cells using vectors that are responsive to miRNA

regulation. A study has shown that by challenging mice with

lentiviral vectors encoding target sequences of endogenous

miRNAs, the efficiency of miRNAs is achieved in sharply segregating the gene expression among different tissues [87].

In another study, analysis of the relationship between

miRNA expression levels and target mRNA suppression

suggests that the suppression depends on a threshold miRNA

concentration, which makes it pertinent to exploit the

miRNA regulatory pathway and to generate vectors that

could rapidly adjust transgene expression in response to

changes in miRNA expression [84]. The properties of

miRNA-regulated vectors should allow for safer and more

effective therapeutic applications [88]. This fast target vali-

dation and easy synthesis of miRNAs are attracting attention from drug developers, as a potential multi-billion dollar

therapeutic platform in the next few years [7].

Metabolic Engineering

The approaches used to improve foreign-protein produc-

tion in various expression systems include strain improve-

ment by mutagenesis and screening and genetic modifica-

tions such as the deletion of proteases from the production

strain, the introduction of multiple copies of expressed

genes, the use of strong promoters, gene fusions to well-

secreted proteins, the use of native signal sequences, and

overexpression of individual endoplasmic reticulum-resident genes [89-92]. The basic idea is for increased productivity,

cost reduction, and for developing new strains with more

specific desirable characteristics such as achieving a more

complete PTMs that could accentuate, diminish or eliminate

the activity of the desired enzyme. This may require a more

comprehensive analysis of the recombinant organism in

terms of its biology, kinetics, physiology and performance.

Metabolic engineering takes strain improvement, from

empirical approach through mutagenesis and selection, to a

more directed improved productivity through the modifica-

tion of specific biochemical(s) or the introduction of new

one(s), via molecular biology, physiology, bioinformatics, computer modeling and control engineering [9-12]. Meta-

bolic engineering comprises a synthesis step that introduces

new pathways and genetic controls; an analysis step to eluci-

date the properties of metabolic reaction networks in their

entirety; and the evaluation of the recombinant physiological

state via metabolic flux determination [11]. The way and the

speed of improving biosystems is greatly changed through

this systems-based analysis as it involves identifying the

reaction and/or transport bottlenecks, thermodynamic feasi-

bility, pathway flux distribution and flux control.

Metabolic engineering has already made impact in drug

discovery through the synthesis of enhanced or novel natural

products and proteins such as carotenoids, ascorbic and lac-

tic acids, xylanases, progestrones, amino acids and novel

precursors to amino acids, biopolymers and chiral chemicals,

extension of substrate range for growth and product forma-

tion, and the detoxification, biodegradation or mineralization

of toxic pollutants [93]. The metabolic engineering approach

in the precursor formation of useful stereochemistry has been

demonstrated in the bioconversion of indene to 1-indenol

and cis-(1S,2R)-indandiol, from which Cis-aminoindanol, a key chiral precursor to the HIV protease inhibitor CRIX-

IVAN, can be derived. A new operon encoding a toluene-

inducible dioxygenase (TID) discovered from Rhodococcus

sp. I24 strain, has shown the capability of converting indene

to 1-indenol and cis-(1S,2R)-indandiol, when the operon is

heterologously expressed in E. coli [94].

Genomics, Transcriptomics, Proteomics, Metabolomics and Fluxomics

The synthesis aspect in metabolic engineering can be

achieved if the genes to be expressed are available, but analysis is more of a problem, due to the the complexity of

the cellular metabolism and the lack of a breakthrough tech-

nology to deal with it [11,95]. A number of powerful tech-

niques have been developed that enable a far more in-depth

analysis of the cellular physiology. These include DNA se-

quencer for genomic analysis, DNA microarray for transcrip-

tomic analysis (simultaneous quantification of all gene tran-

scripts in a cell), two-dimensional gel electrophoresis, pro-

tein microarray and protein function microarray for proteo-

mic analysis (simultaneous quantification of a large number

of proteins in a cell), gas chromatography (GC), high per-formance liquid chromatography (HPLC), nuclear magnetic

resonance (NMR), direct injection mass spectrometry (MS),

or Fourier transform infrared (FT-IR) and Raman spectros-

copy for metabolomic analysis (analysis of the intracellular

metabolite levels), advanced fermentation technology with

on-line control and monitoring, and bioinformatics (includ-

ing mathematical models for analysis of pathway structures

and control of pathway fluxes) [95-97]. Advances in se-

quencing and DNA replication technologies have made it


possible to sequence the entire genomes of many organisms.

Genomes of more than 170 microorganisms have been com-

pletely sequenced and more than 190 sequencing projects

were active in July 2004 [98]. The completion of these se-

quencing projects coupled with rapid development of ge-

nome-derived technologies will spur the effort towards link-

ing the global gene expression analysis with cell physiology.

Of greater impact in this effort is the development of protein

microarrays and protein function arrays which have been suggested to have greater diversity of assays than DNA mi-

croarrays. A novel type of protein array has been developed

where the recombinant proteins are bound to the surface

without possible losses of functions. The first yeast proteome

chip investigating protein-protein interactions and lipid-

binding; and a human proteome chip composed of 5000 pro-

teins have been reported [99].

Another indispensable area in metabolic engineering for

direct pathway modification and strain analysis is me-

tabolomics analysis. Metabolomics offers the unbiased abil-ity to differentiate organisms or cell states based on metabo-

lite levels that may or may not produce visible pheno-

types/genotypes. To understand the global cellular functions

at multi-controlling steps, it is imperative to carry out com-

bined analysis of transcriptome, proteome and/or me-

tabolome simultaneously [100]. In comparing two different

strains of E. coli, it has been established through metabolic

flux, NMR/MS and Northern blot analysis, that the glyoxy-

late shunt, the TCA cycle, and acetate uptake by acetyl-CoA

synthetase are more active in E. coli strain BL21 than in

JM109. This has resulted in the differences in the glucose metabolism and acetate excretion. Upon closer examination

with microarrays and time course Northern blot, it is found

that not only the glyoxylate shunt, the TCA cycle and the

acetate uptake are different, the other metabolic pathways

such as gluconeogenesis, anaplerotic sfcA shunt, ppc shunt,

glycogen biosynthesis, and fatty acid degradation, are also

active differently in the two strains [101].

Bioinformatics

The advent of bioinformatics through genome databases,

protein databanks and other databases containing detailed

information about biological systems has changed the land-scape of biological and bioengineering research. This rapid

growth of bioinformatics databases and pattern discovery

methods provides a powerful means of achieving the goals

of metabolic engineering. The combination of computational

biology and expression-based analysis of large amounts of

sequence information emerge as indispensable tools for gene

discovery and characterization. The paradigm has changed

from ‘vertical’ analysis of the role(s) of one or a few genes

to ‘horizontal’ holistic approaches, studying the function of

many or even all of the genes of an organism simultaneously

[96]. This requirement, coupled with the rapidly increasing database size necessitates pattern recognition algorithm for

association formation, feature extraction, and identifying

homogeneous subsets of data with similar characteristics or

dominant discriminating characteristic that can be used for

sequence identification, function assignment or process di-

agnosis [102]. Several pattern recognition algorithms have

emerged which has proven effective in identifying patterns

across all data sets. These include Principal Component

Analysis, Cluster Analysis, Mean Hypothesis Testing, multi-

resolutional scale analysis by Wavelet Transforms, Decision

Trees, and Artificial Neural Networks.

Gene prediction or gene finding is the most important

step to understand the genomic species once it has been se-quenced [103]. The individual genes can be algorithmically

identified from the stretches of sequence, usually genomic

DNA; while the biochemical function of a gene can be de-

duced by comparing the DNA sequence with the sequences

of genes of known function in the databases. A facility

where researchers can interactively scan for recurring pat-

terns in the sequence, or investigate reading frames, variable

regions, positions of particular codons could facilitate quick

overview of the sequence features [104]. New computer

tools may prove indispensable to compose genetic data at all

levels of biological organization - from gene to population,

species and ecosystems - for multiple purposes, including gene conservation. Similarly, structural genomics projects

have begun to produce protein structures with unknown

function. Such progress requires accurate, automated predic-

tors of protein function to be developed if all these structures

are to be properly annotated in reasonable time. Algorithms

that can align more than two sequences (i.e., multiple align-

ments) can help elucidate phylogenetic relationships within

protein families, thus providing new insight into the evolu-

tion of a protein and its potential utility [105,106]. Identify-

ing the interface between two interacting proteins provides

important clues to the function of a protein and can reduce the search space required by docking algorithms to predict

the structures of complexes. An increasingly popular ma-

chine-learning approach, the support vector machine (SVM),

has been applied for protein–protein binding sites prediction

with high accuracy whilst avoiding over-fitting, using the

profiles of spatially and sequentially neighbouring sequences

and also sequence neighbours of a target residue. SVM have

also found applications in gene expression classification,

protein classification, protein fold recognition and prediction

of protein solvent accessibility, -edge strands, single nu-

cleotide polymorphisms, protein secondary structure, protein quaternary structure and T-cell epitopes [106].

Another important area is in protein–protein interfaces

and development of methods for predicting protein interface

residues. The side chains of the amino acids, owing to a

large extent to their different physical properties, have char-

acteristic distributions in interior/surface regions of individ-

ual proteins and in interface/non-interface portions of protein

surfaces that bind proteins or nucleic acids. These distribu-

tions have important structural and functional implications

[107,108]. Interface prediction methods rely on a wide range of sequence, structural and physical attributes that distinguish

interface residues from non-interface surface residues. The

input data are manipulated into either a numerical value or a

probability representing the potential for a residue to be in-

side a protein interface. Accurate methods have been devel-

oped for predicting the solvent accessibility of amino acids

from a protein sequence and for predicting interface residues

from the structure of a protein-binding or DNA-binding pro-


tein [107]. Satisfactory predictions for complex-forming

proteins that are well represented in the Protein Data Bank

have been achieved, but less so for the under-represented

ones. Efforts are reportedly being made in building structural

models for multi-component structural complexes [108].

High Throughput Bioprocess Development

With greater need for rapid sampling and accurate infor-

mation on the interactions between biosystems and the bio-process operations, microfabrication and array-based testing

could revolutionize the drug discovery process. Miniaturized

analytical devices could reduce reagents and sample con-

sumption, and improve the analytical speed by reducing the

time required by running several analyses in parallel [109].

In combination with optical sensor technology, low-cost mi-

crobioreactor is relevant to investigate biological kinetics

and for high-throughput evaluation of the operational or nu-

tritional parameters on cell growth and product formation in

a systematic and statistically significant manner [110,111]. A

multiplexed microbioreactor system with a working volume

of 150 μl for simultaneous operation of up to eight micro-

bioreactors has been reported (Fig. 1) [112]. The reactors,

fabricated of poly(methyl methacrylate) (PMMA) and

poly(dimethylsiloxane) (PDMS), include miniaturized mo-

tors for magnetic stirring of the reactors, and optic sensors

for measuring the fermentation parameters. Optical density is

determined with a transmittance measurement through the

reactor chamber, and in-situ measurements of dissolved oxy-

gen and pH are obtained with fluorescence lifetime sensors

embedded in the bottom of the reactor chambers. The multi-

plexed microbioreactor system monitors and records the

process parameters in real time for each microbioreactor.

Parallel batch culture of E. coli fermentation data of cell

growth, DO, and pH compare favorably with microbial fer-

mentations undertaken with the same strain and under the

same conditions in multiple bioreactor systems at the bench

scale. In another system, a well-mixed, 150 μl, membrane-

aerated microbioreactor run in chemostat mode reach a

steady state condition at which E. coli cell biomass produc-

tion, substrates and the product concentrations have been

reported to remain constant. The reactor is fed by a pressure-

driven flow of fresh medium through a microchannel. Che-

motaxisial back growth of bacterial cells into the medium

feed channel is prevented by local heating. Using

poly(ethylene glycol) (PEG)-grafted poly(acrylic acid)

(PAA) copolymer films, the inner surfaces of PMMA and PDMS of the reactor wall are modified to generate bio-inert

surfaces resistant to non-specific protein adsorption and cell

adhesion. These modified surfaces effectively reduce wall

growth of E. coli for a prolonged period of cultivation [113].

An integrated array of microbioreactors has also been

developed, leveraging on the advantages of microfluidic in-

tegration to deliver a disposable, parallel bioreactor in a sin-

gle chip, rather than on robotically multiplexing independent

bioreactors. The system offers small scale bioreactor arrays

with the capabilities of bench scale stirred tank reactors. The

microbioreactor with 100 μl working volume, comprise a

Fig. (1) (a) Multiplexed microbioreactor system. The ‘‘Sixfors’’ bioreactor system containing six bench-scale reactors (Infors, Switzerland). (b) Fermentation data obtained with the Sixfors. (c) The multiplexed system with four stirred microbioreactors and an integrated microbiore-actor cassette. (d) Fermentation data obtained with the multiplexed microbioreactor system [112] (Reproduced by permission of The Royal Society of Chemistry).


peristaltic oxygenating mixer and microfluidic injectors (Fig.

2). These integrated devices are fabricated in a single chip

and can provide a high oxygen transfer rate (kLa 0.1 s-1)

without introducing bubbles, and closed loop control over

dissolved oxygen and pH (± 0.1). The system reportedly

could support eight simultaneous E. coli fermentations to

cell densities greater than 13 gDW/L comparable to that

achieved in a 4 L bench scale stirred tank bioreactor. This is

more than four times higher than cell densities previously achieved in microbioreactors. Bubble free oxygenation per-

mitted near real time optical density measurements could be

used to observe subtle changes in the growth rate and infer

changes in the state of microbial genetic networks [114].

Rational Drug Design

High-throughput screening (HTS) involves screening of

thousands to millions of compounds to identify target or lead

compound with useful biological activities, and accessing the

libraries of pharmaceutical and chemical companies. Such

technique of blind-screening of millions of compounds in the

lab and hoping for a hit or a lead has increasingly be seen instead as an irrational approach. Rational drug design which

is synonymous with structure-based design, draws the em-

phasis away from traditional random screening. It involves a

logical, calculated approach, which may include ligand-

based approach to discovery [2,115]. It relies heavily upon

computer modeling to modify an existing drug or design a

new drug which will interact with selected molecular target

important in disease progression. In silico methods are be-

coming more efficient as they allow scientists to hone in on

and manipulate specific molecular structures of interest. A

pre-requisite is the three dimensional structure of the drug’s

target be known to ease the finding of the molecules that

would interact more efficiently in an active protein site, and

subsequently assist the chemists to design more efficient

drugs [2,116]. Targets are normally proteins such as specific

enzymes or receptors for hormones or ligand that would modify the target activity. An example being the activity of

retroviral reverse transcriptase as an effective AIDS thera-

peutic agent. Predictive computer modeling software allows

generation of a likely 3D structure from the amino acid pri-

mary sequence. This however must be complemented by X-

ray crystallography to determine the exact 3D structure.

Once the 3D structure of the target protein has been resolved,

molecular modeling software facilitates rational design such

as a small ligand capable of fitting into a region of an en-

zyme’s active site [2].

The development of combinatorial libraries, through

techniques capable of generating large numbers of novel

synthetic chemicals, coupled with high-throughput screening

methods and the use of sophisticated knowledge-based ap-

proaches to drug discovery is becoming routine [2]. The dis-

covery informatics software for virtual HTS (vHTS) will

continue to play a vital role in rational drug design [115]. It

has been suggested that without computer modeling, identi-

Fig. (2). Microbioreactor array module. (a) Four reactors integrated into a single module. (b) Cross-section of a microbioreactor showing the peristaltic oxygenating mixer tubes and fluid reservoir with pressure chamber. (c) Top view of a microbioreactor showing optical sensors and

layout of peristaltic oxygenating mixer and fluid injectors. Growth well is 500 mm deep, with a 100 μl working volume. (d) Cross-section

showing the fluid injector metering valves [114] (Reproduced by permission of The Royal Society of Chemistry).


fication of a potent drug would require screening of hun-dreds of thousands of candidates, taking up to 10 years or so,

costing hundreds of millions dollars. Computer modelling

saves time and cost as the discovery can be made with a

software, and fewer compounds to be prepared or modified

to yield a highly effective drug; as compared to the cost in

setting up an experimental HTS laboratory and developing

assays to discover a compound [2,115]. Ironically, one of the

major issues facing pharma and biotechs sector today is the

lack of innovation. This downward spiral has been attributed

among others to the heavy investment in computer-assisted

drug design, in building chemical libraries and in high-throughput screening at the expense of hiring innovative

chemists and biologists [117]. There are challenges in gener-

ating high quality protein crystals to facilitate X-ray analysis,

as NMR can only determine 3D structure of small proteins

[2]. The crystal structure does not always accurately depict

how a molecule will behave in vivo; and the medicinal chem-

ists also often find it difficult to develop new structures for

the “rational” approach [116]. The “omics” technology cou-

pled with efficient and effective “knowledge and disease

management” strategies should offer new opportunities for

achieving rationality in drug design. In addition, much drug

discovery and development data requires the time depend-ence of biological responses, which means collecting the

data at an infinite number of points, and employing time-

series methods to give a clearer understanding of biological

processes. With this new network biology era, it becomes pertinent for quantitative description of all the cellular com-

munication networks and how they integrate. For these, vali-

dation of the networks through statistics to provide estimates

of the robustness of the parameters and network structures;

and identification and confirmation of the genetic regulation

mechanism through fundamental genetics and biochemistry,

are vital [116].

Integrated Platform

Current research on chemical and pharmaceutical devel-

opment and manufacturing for integrated systems focuses on advanced analytical and control techniques, computational

methods for process invention and optimization and knowl-

edge management. High-throughput microscopy and imag-

ing analysis are becoming increasingly important with the

development of fluorescence tagging, live cell experimenta-

tion, image acquisition and processing and computer soft-

ware that brings all together. In pharmacotherapy, where

there is a greater need to observe the changes in real-time, a

microfluidic technology has been developed for

highthroughput live-cell screening, with fluidic control tech-

niques for kinetic studies, changes of media, changes of

drugs or flow mixing, under microscopic scrutiny [118]. There is an increasing trend towards integration of in situ

(on-line) spectroscopic measurements (particularly of reac-

tions), real-time analysis of the spectroscopic signals, and

Fig. (3). Biopharmaceuticals production considerations in a nut-shell (Modified from [120]).


feedback control to feeds, and dosing units in order to

achieve desired reaction rates or selectivity. This is done

with the implementation of chemometrics or multivariate

statistical analysis for elucidating pertinent chemical infor-

mation from various process analytical measurements [118].

Molecular imaging has become useful for drug discovery as

there is a greater interest in understanding the mechanisms at

the gene and molecular level. A new technology STORM

(sub-diffraction limit imaging by Stochastic Optical Recon-struction Microscopy) has been developed where optical

image is built through the orchestration of photon emissions

of individual, switchable fluorescent molecules with molecu-

lar specificity for intracellular details. Another technique,

MIMS (Multi-isotope Imaging Mass Spectrometry) takes

advantage of the existence of stable non-toxic isotopes such

as 15N. Applications include in the pulse chase, small mole-

cule drug target interaction and tracking the lineage of trans-

planted stem cells [119].

CONCLUSIONS

The ultimate aim of biopharma development is to im-prove the quality of life and to extend longevity. The quest

for new drugs is never ending, as is the need to understand

disease causes beyond the symptoms. The rapid emergence

of new technologies is revolutionizing the biopharma in-

dustry. As shown in Fig. (3), the approach in the develop-

ment of biopharmaceuticals require multi-pronged strategies.

Promising among these are the development of molecular

diagnostic technologies to elucidate, evaluate and monitor

diseases, vaccine technology principally the DNA-based

viral vaccine, and the high-throughput screening platform

with real-time monitoring and analysis. The future for bio-pharmaceuticals production is indeed extremely bright and

offers an unprecedented opportunity.

ACKNOWLEDGEMENTS

The authors would like to thank the Universiti Teknologi

Petronas for the financial support given to M.A. Abdullah to

present the paper at the First International Conference on

Drug Design and Discovery, Dubai, 4-7th February, 2008.

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Received: March 18, 2008 Revised: April 20, 2008 Accepted: April 21, 2008

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